In FY12, we have continued to focus primarily on elucidating the tumor cell-autonomous components of the switch in activity of TGF-beta from tumor suppressor to pro-progression factor. Our main experimental platform is a xenograft model of breast cancer progression based on the MCF10A human breast epithelial cell line. We have previously demonstrated that TGF-beta switches from tumor suppressor to pro-progression factor in this model, and the high degree of genetic relatedness between the different cell lines of the progression series gives us an exceptionally high signal-to-noise system in which to address mechanisms underlying the TGF-beta switch. Our work in FY12 has focused in three main areas:1. THE ROLE OF TGF-BETA IN REGULATING CANCER STEM CELL DYNAMICS. During FY12 we have continued to validate and employ a novel functional imaging approach for the identification of the putative cancer stem cell (CSC) population. Our CSC reporter responds to the presence of the stem cell master transcription factors Oct4 and Sox2, and marks cells that are enriched for CSC activities, including the ability to divide asymmetrically, to resist cell killing by conventional chemotherapeutics, and to initiate tumorigenesis and metastasis in vivo. We have shown that the hierarchical organization of the breast cancer models we have examined is more pronounced in vivo than in vitro. We are exploiting this reporter using a variety of approaches including timelapse fluorescence microscopy in vitro and confocal microscopy on tumors ex vivo to address factors that regulate CSC localization, plasticity and behavior, and to screen for therapeutics that target the CSC population. A major focus is the role of TGF-beta superfamily members in CSC biology. To extend this approach from transplanted to spontaneously arising tumors, we have successfully generated transgenic mice expressing the stem cell reporter. Understanding how CSCs are regulated will be critical to development of more effective cancer therapies as these cells are largely resistant to existing therapeutic approaches, leading ultimately to relapse.2. INSIGHTS INTO TGF-BETA-MEDIATED EFFECTS ON TUMORIGENESIS FROM GENOMIC APPROACHES. TGF-beta antagonists are being developed as cancer therapeutics. However, the complex role of TGF-beta in cancer progression makes it imperative to avoid treating patients whose tumors still have intact tumor suppressive responses to TGF-beta. Published TGF-beta response signatures have not distinguished the tumor suppressive responses from the neutral or tumor promoting responses. Using the MCF10-based model of human breast cancer progression, we applied genome-wide chromatin immunoprecipitation and transcriptomic approaches to specifically dissect out a core gene signature that is associated with TGF-beta-mediated tumor suppression. In a meta-analysis of more than 1300 human breast cancers, high expression of this signature associated with good distant metastasis-free survival in women with estrogen receptor positive (ER+) breast cancer, suggesting that the tumor suppressor effects of TGF-beta are still active and affecting disease outcome in a subset of patients. We are probing the underlying biological and molecular mechanisms, and have identified an important role for TGF-beta-induced differentiation in tumor suppression. We are also addressing effects of TGF-beta on the miRome in the same MCF-10-based model system to build an integrated molecular mechanistic picture of the changing role of TGF-beta in breast cancer progression. 3. DEVELOPMENT OF TOOLS TO IDENTIFY AND QUANTITATE NON-CANONICAL TGF-BETA SIGNALING IN HUMAN CLINICAL SAMPLES. Recent data from other labs has suggested that TGF-beta signaling via non-canonical mechanisms is associated with tumor promoting effects of TGF-beta, while canonical signaling is critical for tumor suppressor effects. Thus activation of non-canonical TGF-beta signaling may cause TGF-beta to switch from tumor suppressor to tumor promoter. Non-canonical signaling mechanisms include the formation of 'mixed Smad' signaling complexes involving both TGF-beta Smads (Smad2/3) and BMP Smads (Smad1/5/8). In situ proximity ligation (Duolink) is a new technique that uses paired antibodies to detect and quantitate protein/protein interactions in frozen or fixed tissue sections. We have generated and validated antibodies for the detection of the mixed Smad complexes, and have successfully developed the first bright-field application of this technique for application to formalin-fixed, paraffin-embedded tissue and thus to archived clinical specimens. We have show that mixed Smad complexes are highly expressed in the embryo but not normal adult tissues, and are reactivated in disease states. We are applying this approach to look at the onset of non-canonical signaling at different stages in cancer progression in pre-clinical and clinical breast cancer material. We are also assessing whether non-canonical TGF-beta signaling might serve as a useful predictive biomarker for TGF-beta antagonist trials (see related project Z1A BC 010881).